P652: MEASURING MINIMAL RESIDUAL DISEASE BEYOND 10-4 THROUGH IGHV LEADER-BASED NEXT GENERATION SEQUENCING IMPROVES PROGNOSTIC STRATIFICATION IN CHRONIC LYMPHOCYTIC LEUKEMIA
Authors: P. Hengeveld, M. van der Klift, P. M. Kolijn, F. Davi, F. Kavelaars, E. de Jonge, S. Robrecht, J. Assmann, L. van der Straten, M. Ritgen, P. Westerweel, K. Fischer, V. Goede, M. Hallek, M.-D. Levin, A. Langerak
Journal: HemaSphere (2022)

Background: The sensitivity of conventional techniques for reliable quantification of minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) is limited to MRD 10-4. Measuring MRD <10-4 could help to further distinguish between CLL patients with durable remission and those at risk of early relapse. Aims: To develop an academically sourced IGHV leader-based next-generation sequencing (NGS) assay for the quantification of MRD in CLL. Methods: The IGHV leader primer set developed by the EuroClonality-NGS working group was used to amplify all IGH rearrangements present in an end of treatment (EOT) DNA pool. The IGH clonal target was amplified in a single-step PCR and subsequent sequencing was performed on the Illumina MiSeq platform. To calculate MRD depth, NGS output was annotated through in the ARResT/Interrogate immunoprofiler. Technical validation of the IGHV leader-based assay was performed on contrived MRD samples, created by serial dilution of pretreatment CLL DNA pooled PBMC DNA from healthy donors. Clinical validation of the IGHV leader-based assay was performed on EOT samples from the CLL11 trial (NCT01010061). Results: In 176/183 (96%) measurements on contrived samples, the CLL-specific rearrangement was detected. The limit of detection and limit of quantitation were estimated at 3.4 [95% CI 1.9-16.0] and 3.8 [95% CI 1.7-8.2] malignant cells per assay, respectivelly. The limit of blank was found to be 0. Linearity was established in the MRD 10-2-10-5 range (r=0.94 [95%CI 0.91-0.96]). Of note, when provided with sufficient DNA input, MRD could even be detected down to MRD 10-6. There was high inter-assay concordance between measurements of the IGHV leader-based NGS assay and allele-specific oligonucleotide quantitative PCR (r=0.92, [95%CI 0.86-0.96]) and droplet digital PCR (r=0.93, [95%CI 0.88-0.96]). In a cohort of 67 patients from the CLL11 trial, using 5μg DNA input and MRD 10-5 as a cut-off, undetectable MRD (uMRD) was associated with superior progression-free survival (PFS) and time to next treatment. Importantly, deeper MRD measurement allowed for additional stratification of patients with MRD <10-4 but ≥10-5. Patients with MRD in this range had a significantly shorter PFS, compared to patients with uMRD <10-5, but significantly longer, compared to patients with MRD ≥10-4 (median PFS: ≥10-4, 10.4 months; <10-4 but ≥10-5, 27.5 months; uMRD <10-5, NR, 4-year PFS rate: ≥10-4, 66.7%; <10-4 but ≥10-5, 25.0%; uMRD <10-5, 7.2%, P<0.001) (Figure 1). Image:Summary/Conclusion: We here present an academically-developed, IGHV leader-based NGS assay for the detection and quantification of MRD in CLL beyond MRD 10-4. The assay has high sensitivity and is quantitative and linear up to MRD 10-5 when using 5μg DNA input. Measurement to MRD 10-6 is feasible, conditional on sufficient DNA input. The deeper MRD measurements enabled by the IGHV leader-based NGS assay resulted in improved stratification of CLL patients following treatment.